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Over the past decade, 5-aminolevulinic acid (5-ALA) has been highlighted as a promising functional feed additive and immunomodulator for improving the general health, immune response, and resistance to disease of livestock and poultry. However, it is very costly to produce 5-ALA using conventional chemical synthesis methods. Classical microbial fermentation fulfills the criteria of environmental friendliness, but the unsatisfactory titers still hinder actual industrial production. This study aimed to develop a solid-state fermentation (SSF) process that can be used to efficiently enrich feed with 5-ALA at a low cost. First, the endogenous 5-ALA synthase was overexpressed in Saccharomyces cerevisiae via integrating a copy of HEM1 gene into the chromosome and introducing a multi-copy plasmid pRS416-HEM1 which constitutively overexpresses HEM1 gene. The resulting strain ScA3 was able to produce 63.82 mg/L 5-ALA in shake-flask fermentation. After process optimization, a titer of 225.63 mg/kg dry materials, exceeding the usual effective dosage reported in animal trials, was achieved within 48 h through SSF of 20 kg feed in a 90-L steel drum. To our knowledge, this is the first report on combining microbial 5-ALA production with SSF in feed processing, which will hopefully promote the application and popularization of 5-ALA in the feed industry.

5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.

The authors wish to notify that the part of the nitrogen assimilationmodel dealing with AmtB-mediated transport of ammonium across the cytoplasmic membrane contains an error. The set of reactions constituting ammonium transport (reactions rAN4, rAN3, rN3 and the external NH3/NH + 4 balance reaction: NH3ex +Hex = NH4ex) does not comply with the detailed balance principle. The net reaction of these four reactions is NH3ex =NH3in. In particular, the product of the forward reaction constants should have equalled that of the backward reaction constants, that is, their ratio should equal 1, while it actually amounts to 3.6 in the model. Exactly for this reason, the model allows for intracellular NH3 accumulation (at most 3.6-fold) and the concomitant occurrence of negative NH3 diffusion. Thus, although the transport process was modelled according to what is known about the mechanism (without explicit reference to the driving force that is still a controversial issue), the reaction constants chosen implicitly caused ammonium transport to become a case of active transport. Consequently, at a kAmtB value of 2000 s−1, the steady-state ratio of the internal/external NH3 concentration in the model was 1.3 when besides active transport also nitrogen assimilation, passive NH3 permeation, G lnK activity, and pH were taken into account (see Table 3). The authors apologize for any confusion this error might have caused and thank Dr D. Molenaar for his critical alertness.