Industrial Systems Metabolic Engineering Of Saccharomyces Cerevisiae Enables Lornithine Production

Baker’s yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways, but endogenous metabolism by itself involves many metabolites of industrial interest and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products and hence strains over-producing certain pathway intermediates can serve as platform cell factories for production of such products. Here, in this thesis, we implemented a Modular Pathway Rewiring(MPR) strategy and demonstrated its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. Study in this thesis represents the first comprehensive study on over-producing an amino acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino acid-derived chemicals.The main results were achieved and present as follows:(1) For L-arginine biosynthesis, L-ornithine is converted to L-citrulline by ornithine carbamoyltransferase(ARG3) in the cytoplasm after L-ornithine is exported from the mitochondria. We fine-tuned ARG3 expression rather than deleting the gene so that L-arginine can still be synthesized at low levels to support growth, and this will also limit any negative regulation of L-arginine on L-ornithine biosynthesis. We weakened ARG3 expression by replacing its native promoter with either the glucose regulated HXT1 promoter or the low activity KEX2 promoter. While strain M1 a produced 24 mg l-1L-ornithine, strain M1 b had a 76% higher titre of 42 mg l-1.(2) After optimization of L-ornithine consumption, we set out to optimize Module 2of the L-ornithine biosynthesis pathway from α-ketoglutarate. To coordinate transport and biosynthesis of L-ornithine we applied three different strategies. First, improving mitochondrial L-ornithine biosynthesis combined with engineering of transporters.Second, improving mitochondrial L-ornithine biosynthesis combined with translocation of L-glutamate biosynthesis to the mitochondria. Last but not least,translocation of the entire L-ornithine biosynthetic pathway to the cytoplasm. The first two strategies involved over-expressing genes of the mitochondrial L-ornithine biosynthetic pathway from L-glutamate. These engineered strategies enable the syntetic yeast cell factory which can produce L-ornithine titre to 192 mg l-1.(3) After efficiently channelling α-ketoglutarate toward L-ornithine, we set out to enhance α-ketoglutarate supply by optimizing Module 3, which involves the conversion of glucose to α-ketoglutarate. However, the optimization of this part is more difficult in S. cerevisiae due to the Crabtree effect. The Crabtree effect hereby compromises carbon flux to the TCA cycle that provides α-ketoglutarate required for L-ornithine biosynthesis. To overcome this problem we evaluated three different strategies. First, over-expression of TCA cycle genes involved in biosynthesis ofα-ketoglutarate. Second, improving consumption of NADH generated in connection with α-ketoglutarate biosynthesis. And third, attenuating glucose uptake rate and hereby reducing over-flow metabolism to ethanol. These engineered strategies enable the syntetic yeast cell factory which can produce L-ornithine titre to 778 mg l-1.(4) S. cerevisiae has the potential to operate the urea cycle: arginase(encoded by CAR1) can degrade L-arginine into urea and L-ornithine. To further increase L-ornithine synthesis and reduce the L-arginine pool, which can cause feed-back inhibition, we also over-expressed CAR1. As expected, over-expression of CAR1 in the strain M1 c M2q M3e(resulting in strain M1 d M2q M3e) enabled a final L-ornithine titre of 1,041 mg l-1, representing a further 34% increase. This final strain has a23-fold improvement in L-ornithine production compared with strain M1 c.