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Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol., (© 2019 John Wiley & Sons, Ltd.)

Many natural drugs use acetyl-CoA as the key biosynthetic precursor. While in eukaryotic chassis host like yeast, efficient biosynthesis of these drugs is often hampered by insufficient acetyl-CoA supply because of its compartmentalized metabolism. Reported acetyl-CoA engineering commonly modifies central carbon metabolism to pull and push acetyl-CoA into cytosol from sugars or redirects biosynthetic pathways in organelles, involving complicated metabolic engineering strategies. We constructed a new biosynthetic system based on a Crabtree-negative yeast, which grew exceptionally on ethanol and assimilated ethanol directly in cytosol to acetyl-CoA (3 steps). A glucose-repressed and ethanol-induced transcriptional signal amplification device (ESAD) with 20-fold signal increase was constructed by rewiring native transcriptional regulation circuits. This made ethanol the sole and fast-growing substrate, acetyl-CoA precursor, and strong biosynthetic pathway inducer simultaneously. The ESAD was used for biosynthesis of a commercial hypolipidemic drug intermediate, monacolin J. A strain producing dihydromonacolin L was firstly constructed and systematically engineered. We further developed a coculture system equipped with this upstream strain and a downstream strain with dihydromonacolin L-to-monacolin J module controlled by a synthetic constitutive transcriptional signal amplification device (CSAD). It produced a high monacolin J titre of 2.2 g/L on ethanol in bioreactor. Engineering glucose-supported and ethanol-repressed fatty acids biosynthesis in the upstream strain contributed more acetyl-CoA for monacolin J and improved its titre to 3.2 g/L, far surpassing other reported productions in yeasts. This study provides a new paradigm for facilitating the high-yield production of acetyl-CoA derived pharmaceuticals and value-added molecules., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)