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Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

Summary The unconventional yeast Pichia kudriavzevii is renowned for its ability to survive at low pH and has been exploited for the industrial production of various organic acids, especially succinic acid (SA). However, P. kudriavzevii can also utilize the di‐ and tricarboxylate intermediates of the Krebs cycle as the sole carbon sources for cell growth, which may adversely affect the extracellular accumulation of SA. Because the carboxylic acid transport machinery of P. kudriavzevii remains poorly understood, here, we focused on studying its SA transportation process from the perspective of mining and characterization of dicarboxylate transporters in a newly isolated acid‐tolerant P. kudriavzevii strain CY902. Through genome sequencing and transcriptome analysis, two JEN family carboxylate transporters (PkJEN2‐1 and PkJEN2‐2) were found to be involved in SA transport. Substrate specificity analysis revealed that both PkJEN proteins are active dicarboxylate transporters, that can effectively import succinate, fumarate and L‐malate into the cell. In addition, PkJEN2‐1 can transport α‐ketoglutarate, while PkJEN2‐2 cannot. Since PkJEN2‐1 shows higher transcript abundance than PkJEN2‐2, its role in dicarboxylate transport is more important than PkJEN2‐2. In addition, PKJEN2‐2 is also responsible for the uptake of citrate. To our best knowledge, this is the first study to show that a JEN2 subfamily transporter is involved in tricarboxylate transport in yeast. A combination of model‐based structure analysis and rational mutagenesis further proved that amino acid residues 392‐403 of the tenth transmembrane span (TMS‐X) of PkJEN2‐2 play an important role in determining the specificity of the tricarboxylate substrate. Moreover, these two PkJEN transporters only exhibited inward transport activity for SA, and simultaneous inactivation of both PkJEN transporters reduced the SA influx, resulting in enhanced extracellular accumulation of SA in the late stage of fermentation. This work provides useful information on the mechanism of di‐/tricarboxylic acid utilization in P. kudriavzevii, which will help improve the organic acid production performance of this microbial chassis., Two JEN2 transporters responsible for the utilization of dicarboxylic acids in P. kudriavzevii were identified and functionally characterized. The PkJEN2‐2 transporter also possesses specific tricarboxylic acid transportation activity. Inactivation of PkJEN transporters can be used as a useful strategy to increase SA titer in P. kudriavzevii.

The 14α-hydroxysteroids have specific anti-gonadotropic and carcinolytic biological activities and can be produced by microbial biotransformation. The steroid 11β-/14α-hydroxylase P-450lun from Cochliobolus lunatus is the only fungal cytochrome P450 enzyme identified to date with steroid C14 hydroxylation ability. Previous work has mainly revealed the 11β-hydroxylation activity of the P-450lun towards cortexolone (RSS) substrate; however, the potential steroid 14α-hydroxylation activity of this enzyme, especially for androstenedione (AD) substrate, has not yet conducted in-depth testing. In this work, we further tested the steroid 14α-hydroxylation activity of the P-450lun towards RSS and AD in the Saccharomyces cerevisiae system. We demonstrated that P-450lun functions as the specific 14α-hydroxylase towards the AD substrate (regiospecificity > 99%); however, it showed a poor C14-hydroxylation regiospecificity (around 40%) for the RSS substrate. In addition, through transcriptome analysis combined with gene functional characterizations, we also identified and cloned the gene for the P-450lun-associated redox partner CPRlun. Finally, through codon optimization, knockout of genes for the side reactions related enzymes GCY1 and YPR1, and increasing copies of the P-450lun and CPRlun, we developed a recombinant S. cerevisiae biocatalyst based on the C. lunatus steroid 14α-hydroxylation system to produce 14α-hydroxysteroids. Initial production of 14α-OH-AD (150 mg/L day productivity, 99% regioisomeric purity, and 60% w/w yield) and 14α-OH-RSS (64 mg/L day productivity, 40% regioisomeric purity, and 26% w/w yield) were separately achieved in shake flasks; these results represent the highest level of 14α-hydroxysteroid production in the current yeast system.

D-glycerate is an attractive chemical for a wide variety of pharmaceutical, cosmetic, biodegradable polymers, and other applications. Now several studies have been reported about the synthesis of glycerate by different biotechnological and chemical routes from glycerol or other feedstock. Here, we present the construction of an Escherichia coli engineered strain to produce optically pure D-glycerate by oxidizing glycerol with an evolved variant of alditol oxidase (AldO) from Streptomyces coelicolor. This is achieved by starting from a previously reported variant mAldO and employing three rounds of directed evolution, as well as the combination of growth-coupled high throughput selection with colorimetric screening. The variant eAldO3-24 displays a higher substrate affinity toward glycerol with 5.23-fold than the wild-type AldO, and a 1.85-fold increase of catalytic efficiency (kcat/KM). Then we introduced an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible T7 expression system in E. coli to overexpress the variant eAldO3-24, and deleted glucosylglycerate phosphorylase encoding gene ycjM to block the consumption of D-glycerate. Finally, the resulting strain TZ-170 produced 30.1 g/l D-glycerate at 70 h with a yield of 0.376 mol/mol in 5-l fed-batch fermentation.

C-2α hydroxylated triterpenoids are a large class of plant secondary metabolites. These compounds, such as maslinic, corosolic and alphitolic acid, have important biological activities against HIV, cancer and diabetes. However, the biosynthesis pathways of these compounds have not been completely elucidated. Specifically, the cytochrome P450 (CYP) enzyme responsible for C-2α hydroxylation was unknown. In this study, a novel CYP enzyme that catalyzes C-2α hydroxylation was identified in Crataegus pinnatifida (Hawthorn) using a metabolic engineering platform. It is a multifunctional enzyme with C-2α oxidase activity on oleanane-, ursane- and lupane-type pentacyclic triterpenoids. In addition, the complete biosynthesis pathways of these three triterpenoids were reconstituted in yeast, resulting in the production of 384, 141 and 23 mg/L of maslinic, corosolic and alphitolic acid, respectively. This metabolic engineering platform for functional gene identification and strain engineering can serve as the basis for creating alternative pathways for the microbial production of important natural products.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a widespread modification of plant natural products (PNPs), which exhibit a wide range of bioactivities, and are of great pharmaceutical, ecological and agricultural significance. However, functional annotation is available for less than 2% of the family 1 UGTs, which currently has 20,000 members that are known to glycosylate several classes of PNPs. This low percentage illustrates the difficulty of experimental study and accurate prediction of their function. Here, a synthetic biology platform for elucidating the UGT-mediated glycosylation process of PNPs was established, including glycosyltransferases dependent on UDP-glucose and UDP-xylose. This platform is based on reconstructing the specific PNPs biosynthetic pathways in dedicated microbial yeast chassis by the simple method of plug-and-play. Five UGT enzymes were identified as responsible for the biosynthesis of the main glycosylation products of triterpenes in Panax notoginseng, including a novel UDP-xylose dependent glycosyltransferase enzyme for notoginsenoside R1 biosynthesis. Additionally, we constructed a yeast cell factory that yields >1 g/L of ginsenoside compound K. This platform for functional gene identification and strain engineering can serve as the basis for creating alternative sources of important natural products and thereby protecting natural plant resources.

With the development of CRISPR/Cas9 technology, a new generation of editing methods that convert specific bases has enabled precise single-base mutations. To date, conversion of cytosine to thymidine and adenine to guanine has been achieved using the cytidine deaminase APOBEC1 and adenosine deaminase (TadA), respectively. However, the base editing efficiency can be unacceptably low in some cell types or at certain target loci. One reason might be the lack of a selective pressure against the survival of nonedited cells. Few studies on ABE in prokaryotes have been reported, probably due to the relatively low editing efficiency of TadA. Improving the editing efficiency is the key for establishing base editing techniques and especially the ABE technologies. In this work, a selective pressure against nonedited cells was implemented to increase the base editing efficiency. First, we fused nCas9 or dCas9 with TadA to compare the editing efficiency of nCas9-TadA and dCas9-TadA fusion complexes in the model prokar...

Improvement in the osmotolerance of Escherichia coli is essential for the production of high titers of various bioproducts. In this work, a cusS mutation that was identified in the previously constructed high-succinate-producing E. coli strain HX024 was investigated for its effect on osmotolerance. CusS is part of the two-component system CusSR that protects cells from Ag(I) and Cu(I) toxicity. Changing cusS from strain HX024 back to its original sequence led to a 24% decrease in cell mass and succinate titer under osmotic stress (12% glucose). When cultivated with a high initial glucose concentration (12%), introduction of the cusS mutation into parental strain Suc-T110 led to a 21% increase in cell mass and a 40% increase in succinate titer. When the medium was supplemented with 30 g/liter disodium succinate, the cusS mutation led to a 120% increase in cell mass and a 492% increase in succinate titer. Introducing the cusS mutation into the wild-type strain ATCC 8739 led to increases in cell mass of 87% with 20% glucose and 36% using 30 g/liter disodium succinate. The cusS mutation increased the expression of cusCFBA , and gene expression levels were found to be positively related to osmotolerance abilities. Because high osmotic stress has been associated with deleterious accumulation of Cu(I) in the periplasm, activation of CusCFBA may alleviate this effect by transporting Cu(I) out of the cells. This hypothesis was confirmed by supplementing sulfur-containing amino acids that can chelate Cu(I). Adding methionine or cysteine to the medium increased the osmotolerance of E. coli under anaerobic conditions. IMPORTANCE In this work, an activating Cus copper efflux system was found to increase the osmotolerance of E. coli . In addition, new osmoprotectants were identified. Supplementation with methionine or cysteine led to an increase in osmotolerance of E. coli under anaerobic conditions. These new strategies for improving osmotolerance will be useful for improving the production of chemicals in industrial bioprocesses.

Both mammalian tensin-like phosphatase 1 [TEP1; also known as phosphatase deleted on chromosome 10 (PTEN) or mutated in multiple advanced cancer 1 (MMAC1)] and Saccharomyces cerevisiae ScTep1p are involved in the phosphatidylinositol pathway. In this study, we identified the Fusarium graminearum locus FGSG_04982.3 (named FgTEP1) as the functional homologue of ScTEP1 in the sensitivity of S. cerevisiae cells to wortmannin, the phosphatidylinositol-3 kinase inhibitor. Deletion of FgTEP1 causes F. graminearum mycelial growth to become sensitive to lithium and reduces the production of conidia. Although conidia lacking FgTEP1 germinate normally, they show reduced germination efficiency in the presence of wortmannin. In addition, we showed that deletion of FgTEP1 reduces the virulence of F. graminearum on wheat. These results indicate that FgTep1p is linked to the phosphatidylinositol-3 kinase signalling pathway in this plant fungal pathogen.

Fusarium head blight (FHB) is a worldwide devastating disease of wheat, caused primarily by species in the Fusarium graminearum (Fg) complex. In this study, we obtained 55 Fusarium isolates from wheat with FHB collected from seven provinces along the north of the Yangtze River. One additional phylogenetic species of Fg complex, Fusarium meridionale, was identified for the first time from China in addition to two known ones, Fusarium asiaticum and F. graminearum. In addition, Fusarium acuminatum, distantly related to Fg complex, was for the first time identified in Northern China. Sensitivities of these isolates to carbendazim were examined and appeared to vary both within and between species. Mycotoxin genotype analyses indicated that F. asiaticum isolates were potential 3-AcDON and NIV mycotoxin producers, while all F. graminearum isolates might be 15-AcDON producers. These findings would provide useful information for developing management strategies for the control of FHB in Northern China.

Fusarium head blight (FHB) is a worldwide devastating disease of wheat, barley and other small grain cereals and caused primarily by Fusarium graminearum. Carbendazim (MBC) is one of the fungicides widely used to control FHB in China. In the present study, we have identified the F. graminearum locus FGSG_04220 as the sequence homolog for Saccharomyces cerevisiae ScSWI6, named FgSWI6 hereafter. Deletion of FgSWI6 causes mycelium of F. graminearum become sensitive to MBC in liquid medium. In addition, deletion of FgSWI6 reduces mycelial growth as well as production and development of conidia. F. graminearum cells lacking FgSWI6 show reduced production efficiency and sizes of perithecia as well as a defect in the production of ascus and ascospore. FgSWI6 is required for the cellulose utilization, lithium tolerance and deoxynivalenol (DON) production of this pathogen. Furthermore, deletion of FgSWI6 significantly attenuates the virulence of F. graminearum on wheat. Therefore, FgSwi6p plays an important role in growth and development of the economically important fungal pathogen F. graminearum as well as its resistance to MBC.